Metagenomic Analysis of Different North American Bat Species and the differing Viromes they contain
Introduction Bats are considered to be incredibly important as hosts for several different zoonoses, such as Ebola, Marbug, Nipah, Hendra, sever acute respiratory syndrome-coronaviurs (SARS-CoV) and rabies (1). Although, the diversity in the viral population among bats has only been partially solved for a few of the nearly 1200 species of bats (1). Effective prediction of future viral zoonoses demands a comprehensive understanding of viral populations within a given species, specifically potential host species, such that the next viral epidemic may be addressed quicker and with more precision. Metagenomics and Viromes Metagenomics is considered to be studies of genetic material across multiple species usually gathered from the environment. There are many different classes of metagenomics, ranging from shotgun sequencing, to deep sequencing, high-throughput screens, comparisons of cDNA libraries and PCR analysis (2) . While there are many different ways to approach the understanding of genetic material across genomes, viruses provide a distinct challenge usually not present in other organisms such as bacteria, archea or classical eukaryotes. They do not contain shared phylogenetic markers, such as 16S RNA or 18S RNA (2). Thus the only way to properly asses genetic diversity of a viral population via an environmental sample is through metagenomic analysis. Viral metagenomes (Viromes) help shed light on viral diversity and evolution as they mutate and travel from host to host (3) . Viral Diversity in Different North American Bats with the same Habitat In past years, animals harvested for meat in Africa either as hunters, farmers or animal handlers and the genetic analysis of these tissues have resulted in significant data depicting potential origins, geographic distributions and the emergence of several important human pathogens (4). Viruses can and often remain dormant in a particular host, although, when transferred across species, can have severely deleterious consequences. Multiple human viral epidemics have stemmed from this very notion such as HIV (chimpanzees) (5) , H1N1 (pigs and birds) (6) and SARS-CoV (Chinese horseshoe bat) (7). Further studies on bat derived coronavirus (CoV) has shed light on the worldwide distribution of differing CoVs within bat species (1). Donaldson et al., conducted a study where a total of 512 samples were collected in a single night from 7 different species of North American bats in an abandoned railroad tunnel in Maryland, USA. In this study, the group was able to identify viral sequences that contained homology to three novel group 1 CoVs, several insect and plant viruses and nearly full-length genomic sequence of two novel bacteriophages (1). Identification of novel coronavirus sequences Next-generation sequencing yielded a total of 611,745 sequence reads that contained one of the six unique bar code sequences and segregated based on these sequences. This resulted in 576,274 trimmed reads, placed into six pools. Each bar code bin was formatted and BLAST searched against other known CoVs. This search resulted in a total of ~45,000 sequence reads from all pools, around 7-8% of the whole. The group then attempted to identify novel CoV sequences within this population. 76 total individual sequence reads contained strong homology to known CoV sequences. Of the three different bats examined, three novel group 1 CoVs were detected in juvenile brown bats, designated ARCoV. These novel CoVs sequences were verified via RT-PCR utilizing 454 sequence reads to design primers that recognized and successfully amplified a 2,540nt fragment. All data collected from Donaldson et al. (1). Detection of Novel Insect and Plant Viruses Multiple contigs from pools 1 through 5 were shown to have strong sequence homology with two related picorna-like viruses of insects. Phylogenetics determined the 1,449nt region showed the novel viruses clustered with other viruses of the Iflaviridae ''family. The group determined that honeybee viruses were the closest family member and percent identity at the amino acid level ranged between 27.7% and 41.9%. This suggests that the honeybee virus was capable of infecting an unknown arthropod, a distinct food group of bats. Two configs from pools 3 and 5 contained regions in 1902nt that displayed homology to the grapevine fleck virus (GFkV). These two configs, intriguingly shared 99.9% likeness although only 56% homology with GFkV. Phylogenetics determined that the virus is most similar to other plant viruses in the ''Tymoviridae ''family and most closely related to the ''Marafivirus ''genus. Other sequence reads from pools 1-5 also contained hits in this region, suggesting a novel plant virus localized to a Maryland area plant species. All data and information collected from Donaldson et al. (1). Identification of Novel Bacteriopphage Sequences Several of the pools contained large contig hits that matched different bacteriophages. Pool 1's hit most matched the enterobacteriophage K1F of the ''Podoviridae family. Total reads from the sequence assembled to 88.3% of the K1F genome. Phylogenetic analysis of the novel pool 1 bacteriophage demonstrated that it showed likeness to T7 bacteriophage family. Pool 5's match was determined to be related to Acyrthosiphon pisum secondary endosybiont 1 (APSE-1) bacteriophage. Total reads from this sequence assembled to cover nearly 55% of the APSE-1 genome. Phylogenetic analysis of this sequence suggests it is most likely a novel bacteriophage of the APSE-like family, most related to APSE-4 and APSE-6. All data and information collected from Donaldson et al. (1) References #Donaldson EF, et al. (2010) Metagenomic Analysis of the Viromes of Three North American Bat Species: Viral Diversity among Different Bat Species That Share a Common Habitat. Journal of Virology 84(24):13004-13018. #http://en.wikipedia.org/wiki/Metagenomics #Anderson NG, Gerin JL, & Anderson NL (2003) Global screening for human viral pathogens. Emerging Infectious Diseases 9(7):768-773 #Wolfe ND, Dunavan CP, & Diamond J (2007) Origins of major human infectious diseases. Nature 447(7142):279-283. #Gao F, et al. (1999) Origin of HIV-1 in the chimpanzee Pan troglodytes troglodytes. Nature 397(6718):436-441. #Fraser C, et al. (2009) Pandemic Potential of a Strain of Influenza A (H1N1): Early Findings. Science 324(5934):1557-1561. #Li WH, et al. (2006) Animal origins of the severe acute respiratory syndrome coronavirus: Insight from ACE2-S-protein interactions. Journal of Virology 80(9):4211-4219.